PROJECT SUMMARY/ABSTRACT Heart failure is a major cause of morbidity and mortality in the population, with the rising incidence of cardiovascular disease contributed to by epidemic increases in the incidence of obesity and associated type 2 diabetes mellitus. Although existing drugs are effective in acutely correcting decompensated heart failure, this continues to be associated with unacceptable rates of rehospitalization and mortality. A new drug combination that adds neprilysin inhibition to the current standard-of-care for heart failure, angiotensin receptor blockade, was approved by the FDA on the basis of extension of the life of patients with reduced cardiac output. However, the very broad action of the peptidase inhibitor in this combination theoretically could be improved, thereby reducing possible side effects and increasing therapeutic effectiveness. The specific enzyme substrate responsible for the beneficial effects of this drug is not yet clear. While it has been assumed to be natriuretic peptides, other peptides are also cleaved, many of which have potential offsetting and/or side effects. Another prominent potentially beneficial substrate of neprilysin is secretin, a peptide hormone with useful effects on cardiac contractility and ejection fraction, coronary perfusion, peripheral vascular resistance, post-cibal satiety, and glucose-sensitive incretin action. There are currently no known drugs that target the secretin receptor, providing these effects. This proposal is designed to develop appropriate assays and to screen for candidates with potential to impact this major public health problem using high throughput screening (HTS). The OBJECTIVE is to identify molecules that enhance signaling at the secretin receptor, either by acting as positive allosteric modulators of secretin action or as direct agonists of this potentially important therapeutic target. This represents a partnership between Dr. Miller, with extensive secretin receptor experience, and Dr. Sergienko, with HTS and chemical genomic expertise. There are three specific aims: (i) Primary and secondary assay implementation and optimization, (ii) HTS using cells expressing human secretin receptor and hit confirmation, and (iii) Hit validation and characterization. The primary assays will focus on intracellular cAMP responses in the CHO-SecR cell line stably expressing wild type human secretin receptor, with the assays run in both positive allosteric modulator mode in the presence of submaximal secretin stimulation and in agonist mode without exposure to secretin. During confirmation, the assays will be repeated with compound concentration- dependency evaluated and using a neuronal cell line that naturally expresses a physiologic density of secretin receptor, NG-108 cells. Parental CHO cells and a CHO cell line expressing the Gs-coupled type 2 vasopressin receptor will be studied as controls to be certain that observed responses are secretin receptor-mediated. Selectivity will be explored using other class B GPCR-expressing cells, and a survey of other signaling events, internalization, cell cytotoxicity, and proliferation will be performed. The goal will be to identify candidates with adequate activity to be tested in pre-clinical systems for proof-of-concept, and ultimately in clinical trials.